Part:BBa_K3408007
Pnar-B0034-phy(ycD)-B0015
Used Pnar(BBa_K3408000), RBS(BBa_B0034), phy(ycD)(BBa_E0040) and terminator(BBa_B0015) to express Phy(ycD) under the control of Pnar. When our Bacillus subtilis was in an anerobic environment, Pnar was activated by FNR so that Phy(ycD) could be expressed. But when our Bacillus subtilis was in an aerobic environment, Pnar could be suppressed and we couldn’t detect more Phy(ycD).
1. Experimental methods
1.1. Construction of the expression vector
The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of phytase and terminator of this device are synthesized by the biotechnology company with 6×His tags added. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-Pnar-phy(ycD).
Plasmid profile
Fig.1. The expression vector of device Pnar-phy(yCD)
1.2.Construction and screening of recombinant engineered bacteria
Using B. subtilis WB800N as the expression host, the secretion expression vector pWB980-DB was transformed by electro-transformation. Inoculate them on LB solid medium coated with 10μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.
1.3.Phytase expression and purification
Set up two groups of experiments:
(1) the control group: recombinant Bacillus subtilis are cultured in an aerobic condition; (2) the test group: recombinant Bacillus subtilis are cultured in an anaerobic condition.
①Inoculate recombinant Bacillus subtilis in 20 mL of LB liquid medium containing 10 μg/mL kanamycin, and cultivate them overnight at 37°C with shaking at 180 rpm.
②Inoculate 2% of the overnight cultured bacteria in 100 mL of LB liquid medium containing 10μg/mL kanamycin, and culture them with shaking at 25°C for 24 hours. The supernatant was collected by centrifugation to obtain the crude enzyme solution, and the pure enzyme solution was obtained after Ni-NAT affinity chromatography and Superdex-75 gel chromatography. The purified protein is subjected to SDS-PAGE gel electrophoresis, western blot to determine phytase expression. And gel chromatography is used to obtain the elution profile of the enzyme after gel purification.
③Western Blot：After SDS-PAGE gel electrophoresis, transfer to membrane (wet transfer: 300 mA, 1 h), seal with 5% skimmed milk powder at 4°C overnight, and add TBST (Tris buffered salt-Tween solution, containing 10 mmol/L), pH 7. 6 Tris-HCl, 150 mmol /L NaCl, 0.05% Tween-20) 1:10 000 diluted His primary antibody, incubate at 37°C for 1 h, wash the membrane with TBST 3 times, 15 min each time; Add His secondary antibody diluted 1:20 000 with TBST, incubate at 37°C for 1 h, wash the membrane with TBST 3 times, 15 min each time; HRP-DAB substrate color kit for color development, use Bio-Rad gel imaging system to take pictures.
1.4.Verification of the effect of phytase on phosphate hydrolysis
①Preliminary identification of engineered bacteria expressing phytase
Pick a single colony of the transformant and inoculate it on a LB solid medium containing calcium phytate, and then observe the hydrolysis circle after culturing in a 37 ℃ incubator for 36 h.
②Preparation of phosphorus standard curve
Configure 50 mmol standard KH2PO4 solution (weigh 0.6804 g KH2PO4 and dilute it to 100 mL with pH5.5 acetate buffer), and then dilute it to 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 mmol/mL nine concentration gradients with deionized water. Take 1 mL of gradient solution and add 1 mL of 5.0mmol/L sodium phytate solution, then add 2 mL of stop solution, measure absorbance at 415 nm, do 3 sets of repeated experiments for each concentration gradient, and average within the standard deviation range. Draw a standard curve with the concentration of inorganic phosphorus as the abscissa and the absorbance value as the ordinate.
③Determination of phytase activity
Add 0.8 mL of sodium phytate to 0.2 mL of diluted enzyme solution. After reacting for 15 min at pH 6.5 and temperature of 37 ℃, add 1 mL of 5% TCA (trichloroacetic acid) to stop enzyme activity reaction, and then add 1 mL of ferrous sulfate-ammonium molybdate coloring solution, determine the content of inorganic phosphorus in visible light at 415 nm, and calculate the specific activity. (Enzyme activity unit definition: Under the conditions of 37°C and pH 6.5, the amount of enzyme that releases 1 μmol of inorganic phosphorus from a 5.0 mmol/L sodium phytate solution per minute is defined as 1 enzyme activity unit (U).)
1.5.Verification of the effect of phytase to dissolve phosphorus and solid lead
①Experimental reagents: sodium phytase solution 1.5mM, phytase solution, 230mg/L PbCl2 solution
②test group:
Set test groups for determination of phytase activity
③Experimental steps: Set up four groups of experiments, with whether to add sodium phytase solution and whether to add phytase solution as variables. When phytase solution or sodium phytase solution is not added, the same amount of ddH2O is used instead. In each group of experiments, 15ml of 230mg/L PbCl2 was added to react for 1h, and then the lead content in the reaction system was determined by dithizone colorimetry.
The specific operation steps are as follows:
First, prepare a lead standard series with lead content of 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0μg, and measure it in the range of 540nm and pH 8.5-pH 11, and draw a standard curve based on the data.
Secondly, take 10ml of the reaction solution in a 100ml separatory funnel, add 2ml 20% ammonium citrate, 1ml 20% hydroxylamine hydrochloride, 2d phenol red indicator, adjust the pH to 8.5-9.0 with concentrated ammonia and add 1ml 10% potassium hydride, shake well. Add 10ml of dithizone chloroform application solution, shake and layer, put the chloroform layer into a clean 10ml colorimetric tube, measure the spectrophotometry at 540nm, and find out the corresponding content from the standard curve.
2.Expected results
2.1.Phytase expression and purification
The control group has no production of phytase, and the test group has production of phytase. The molecular weight that can be determined by SDS-PAGE analysis of the expressed enzyme is 45 KD, and the protein can be determined as phytase by Western blot.
Fig.2. Expected results: SDS-PAGE image of predicted expression product.
2.2.Verification of the effect of phytase on phosphorus hydrolysis
①Preliminary screening of strains
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